h163a mutant Search Results


h163a mutant  (GenScript corporation)
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GenScript corporation h163a mutant
Mpro is an obligate homodimeric cysteine protease (PDB 7BB2). (a) Each monomer can be broken up into three regions: Domain I (residues 1-101; yellow/orange); Domain II (102-184; light violet/magenta); and Domain III (201-301; pale green/forest green). (b) The active site in each monomer is created from the interface between Domains I and II, whereby the catalytic dyad’s H41 and C145 are derived from Domains I and II, respectively. In the WT structure, the active-site cysteine (C145) is located ∼12 Å from C117, the cysteine involved in the disulfide bond in the <t>H163A</t> Mpro structure. (c) Surface representation of the WT Mpro with a focus on the active-site cleft. The enzyme’s S2 to S4 pockets are denoted by the black line. The key residue of interest, H163, is located in a pocket laterally connected to this active site groove (denoted by “*”). (d) The surface representation from (c) is rotated 90° counterclockwise to show this H163 lateral pocket from a head-on perspective. Side chains that make up the lateral pocket and the catalytic dyad are rendered as cylinders in both (c) and (d). All molecular representations in this paper were generated in CCP4MG.
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Mpro is an obligate homodimeric cysteine protease (PDB 7BB2). (a) Each monomer can be broken up into three regions: Domain I (residues 1-101; yellow/orange); Domain II (102-184; light violet/magenta); and Domain III (201-301; pale green/forest green). (b) The active site in each monomer is created from the interface between Domains I and II, whereby the catalytic dyad’s H41 and C145 are derived from Domains I and II, respectively. In the WT structure, the active-site cysteine (C145) is located ∼12 Å from C117, the cysteine involved in the disulfide bond in the H163A Mpro structure. (c) Surface representation of the WT Mpro with a focus on the active-site cleft. The enzyme’s S2 to S4 pockets are denoted by the black line. The key residue of interest, H163, is located in a pocket laterally connected to this active site groove (denoted by “*”). (d) The surface representation from (c) is rotated 90° counterclockwise to show this H163 lateral pocket from a head-on perspective. Side chains that make up the lateral pocket and the catalytic dyad are rendered as cylinders in both (c) and (d). All molecular representations in this paper were generated in CCP4MG.

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: Mpro is an obligate homodimeric cysteine protease (PDB 7BB2). (a) Each monomer can be broken up into three regions: Domain I (residues 1-101; yellow/orange); Domain II (102-184; light violet/magenta); and Domain III (201-301; pale green/forest green). (b) The active site in each monomer is created from the interface between Domains I and II, whereby the catalytic dyad’s H41 and C145 are derived from Domains I and II, respectively. In the WT structure, the active-site cysteine (C145) is located ∼12 Å from C117, the cysteine involved in the disulfide bond in the H163A Mpro structure. (c) Surface representation of the WT Mpro with a focus on the active-site cleft. The enzyme’s S2 to S4 pockets are denoted by the black line. The key residue of interest, H163, is located in a pocket laterally connected to this active site groove (denoted by “*”). (d) The surface representation from (c) is rotated 90° counterclockwise to show this H163 lateral pocket from a head-on perspective. Side chains that make up the lateral pocket and the catalytic dyad are rendered as cylinders in both (c) and (d). All molecular representations in this paper were generated in CCP4MG.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Derivative Assay, Residue, Generated

(a) The general fold of Mpro is conserved when comparing the WT (grey; PDB 7TGR) and H163A mutant structures in complex with the covalent inhibitor GC376 (PDB 8DD6). Cα RMSD values were calculated using Chimera. Only one monomer is depicted as the other monomer comprising the dimer is crystallographically identical. (b) The pose of GC376 is also nearly identical between the WT (grey) and H163A mutant structures, although there are slight differences in the ring puckering and rotameric conformation of some inhibitor moieties, particularly the phenyl ring of GC376. These changes are supported by 2F o -F c density at 1.2 σ and can be attributed to the slightly different inhibitor-enzyme interactions between the two structures ( Supplementary Fig. 1 ). (c) The carbonyl on the γ-lactam moiety makes a hydrogen bond with the H163 imidazole ring in the WT enzyme (grey). Upon mutation to alanine, a water molecule compensates for the loss of the imidazole ring by making hydrogen bonds with GC376 and the backbone carbonyl of M165. This is supported by 2F o -F c density at 1.0 σ.

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: (a) The general fold of Mpro is conserved when comparing the WT (grey; PDB 7TGR) and H163A mutant structures in complex with the covalent inhibitor GC376 (PDB 8DD6). Cα RMSD values were calculated using Chimera. Only one monomer is depicted as the other monomer comprising the dimer is crystallographically identical. (b) The pose of GC376 is also nearly identical between the WT (grey) and H163A mutant structures, although there are slight differences in the ring puckering and rotameric conformation of some inhibitor moieties, particularly the phenyl ring of GC376. These changes are supported by 2F o -F c density at 1.2 σ and can be attributed to the slightly different inhibitor-enzyme interactions between the two structures ( Supplementary Fig. 1 ). (c) The carbonyl on the γ-lactam moiety makes a hydrogen bond with the H163 imidazole ring in the WT enzyme (grey). Upon mutation to alanine, a water molecule compensates for the loss of the imidazole ring by making hydrogen bonds with GC376 and the backbone carbonyl of M165. This is supported by 2F o -F c density at 1.0 σ.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

(a) The apo H163A Mpro structure contains a disulfide bond between the active-site nucleophile, C145, and the previously distant cysteine C117 . This disulfide bond is not completely formed as there is 2F o -F c density at 1.2 σ that supports an alternate, non-disulfide-bonded conformation of C117. The side chain of N28 also takes on two conformations, one seen in both WT (grey) and mutant structures and the other only seen in the mutant. There is a concomitant structural change between the formation of the disulfide bond and the rotation of the N28 side chain as the N28 side chain in its WT conformation sterically hinders the disulfide bond from forming. The beta strand containing the active-site nucleophile can take on two conformations depending on whether or not the disulfide bond is present. (b) When the disulfide bond between C145 and C117 is formed, the C145 beta strand runs antiparallel to the C117 beta strand (2F o -F c density shown at 1.2 σ). (c) When the disulfide bond is broken, the C145 beta strand relaxes to a second conformation (F o -F c density shown at 4.0 σ), aligning almost exactly to the WT conformation of the strand when the structures are superimposed. The flexibility in the loop N-terminal to the C145 beta strand (K137-G143) allows for this relaxation to occur. Despite there being crystallographic evidence for both conformational states, only the disulfide-bonded conformation was modelled as density corresponding to the second conformation could not be accurately modelled with only one alternate conformation. (b) and (c) depict the two conformations of chain B of the H163A structure.

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: (a) The apo H163A Mpro structure contains a disulfide bond between the active-site nucleophile, C145, and the previously distant cysteine C117 . This disulfide bond is not completely formed as there is 2F o -F c density at 1.2 σ that supports an alternate, non-disulfide-bonded conformation of C117. The side chain of N28 also takes on two conformations, one seen in both WT (grey) and mutant structures and the other only seen in the mutant. There is a concomitant structural change between the formation of the disulfide bond and the rotation of the N28 side chain as the N28 side chain in its WT conformation sterically hinders the disulfide bond from forming. The beta strand containing the active-site nucleophile can take on two conformations depending on whether or not the disulfide bond is present. (b) When the disulfide bond between C145 and C117 is formed, the C145 beta strand runs antiparallel to the C117 beta strand (2F o -F c density shown at 1.2 σ). (c) When the disulfide bond is broken, the C145 beta strand relaxes to a second conformation (F o -F c density shown at 4.0 σ), aligning almost exactly to the WT conformation of the strand when the structures are superimposed. The flexibility in the loop N-terminal to the C145 beta strand (K137-G143) allows for this relaxation to occur. Despite there being crystallographic evidence for both conformational states, only the disulfide-bonded conformation was modelled as density corresponding to the second conformation could not be accurately modelled with only one alternate conformation. (b) and (c) depict the two conformations of chain B of the H163A structure.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

In addition to the local restructuring of the active site, structural changes are also seen distally in Domain I. An NOS bridge between C22 and K61 is captured in chain B (a) (2F o -F c density shown at 1.2 σ) but not in chain A (b) (2F o -F c density shown at 1.0 σ). (c) This structural asymmetry is also seen when comparing the N-termini of the two monomers, where 2F o -F c density is only seen for the N-terminus of chain B (shown at 1.4 σ) but not chain A. (d) When comparing the positions of the N-termini between the WT and H163A mutant, the four most N-terminal residues are drastically rotated approximately 90° to fit into an alternate pocket. This is due to the movement of the F140 loop in the H163A mutant structure, which occupies the space previously held by the WT N-terminus. There is no density to support a single conformation of the N-terminus in the other protomer.

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: In addition to the local restructuring of the active site, structural changes are also seen distally in Domain I. An NOS bridge between C22 and K61 is captured in chain B (a) (2F o -F c density shown at 1.2 σ) but not in chain A (b) (2F o -F c density shown at 1.0 σ). (c) This structural asymmetry is also seen when comparing the N-termini of the two monomers, where 2F o -F c density is only seen for the N-terminus of chain B (shown at 1.4 σ) but not chain A. (d) When comparing the positions of the N-termini between the WT and H163A mutant, the four most N-terminal residues are drastically rotated approximately 90° to fit into an alternate pocket. This is due to the movement of the F140 loop in the H163A mutant structure, which occupies the space previously held by the WT N-terminus. There is no density to support a single conformation of the N-terminus in the other protomer.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

Many of the structural differences seen between the apo WT and apo H163A Mpro structures can be attributed to the movement of the F140 side chain. (a) F140 is normally found in an “in” conformation within the core of the enzyme (WT structure is in grey), where it is stabilized by a face-to-face π-stacking interaction with the side chain of H163. When the H163 side chain is mutated, F140 flips to an energetically favored “out” conformation in an ∼14 Å motion to situate itself close to the C-terminus of the other monomer. (b) The “out” conformation in the H163A mutant results in the formation of a new hydrogen-bonding network in the space previously occupied by the F140 side chain (2F o -F c density shown at 1.3 σ). This network is formed from two new water molecules (W73 and W179) and the side chains of Y118, Y126, S147, and H172. (c) A hypothetical structural rearrangement of this F140 pocket is shown with arrows indicating the motion of these side chains from their start (WT; grey) to end (H163A) conformations.

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: Many of the structural differences seen between the apo WT and apo H163A Mpro structures can be attributed to the movement of the F140 side chain. (a) F140 is normally found in an “in” conformation within the core of the enzyme (WT structure is in grey), where it is stabilized by a face-to-face π-stacking interaction with the side chain of H163. When the H163 side chain is mutated, F140 flips to an energetically favored “out” conformation in an ∼14 Å motion to situate itself close to the C-terminus of the other monomer. (b) The “out” conformation in the H163A mutant results in the formation of a new hydrogen-bonding network in the space previously occupied by the F140 side chain (2F o -F c density shown at 1.3 σ). This network is formed from two new water molecules (W73 and W179) and the side chains of Y118, Y126, S147, and H172. (c) A hypothetical structural rearrangement of this F140 pocket is shown with arrows indicating the motion of these side chains from their start (WT; grey) to end (H163A) conformations.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Mutagenesis

Free-energy surfaces describing the transitions from state A (WT conformation) to state B (conformation closer to H163A crystal structure) in the WT (a) , H163A (c) , and F140A model (e) are shown. The surfaces were explored for the changes in two CVs, distance between Cα atoms of amino acids at 140 and 163 positions and the sidechain dihedral rotation of N28. The resultant surfaces are depicted in a red to blue spectrum that correspond to high- and low-energy structures, respectively, and the states along the path are marked. The comparison of the 1D free energy profiles corresponding to the minimum energy paths connecting states A and B in the WT, H163A, and F140A models are shown in (b) . The superimposed structures of the H163A crystal structure against the initial and the final states from the metadynamics simulation of H163A model are described in (d) . In the initial state of the H163A model, the side chain of F140 (green cyan) was present in the ‘in’ conformation whereas, at the end of the simulation, the active site loop was dislocated and the F140 sidechain was exposed to the surface – a conformation similar (light cyan) to that seen in the mutant crystal structure. The sidechain rotation of N28 (purple) is also shown. (f) Free-energy profile for the N28A model shows a “free fall” from state A to state B due to a lack of any significant barrier in its path.

Journal: bioRxiv

Article Title: The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

doi: 10.1101/2022.12.16.520794

Figure Lengend Snippet: Free-energy surfaces describing the transitions from state A (WT conformation) to state B (conformation closer to H163A crystal structure) in the WT (a) , H163A (c) , and F140A model (e) are shown. The surfaces were explored for the changes in two CVs, distance between Cα atoms of amino acids at 140 and 163 positions and the sidechain dihedral rotation of N28. The resultant surfaces are depicted in a red to blue spectrum that correspond to high- and low-energy structures, respectively, and the states along the path are marked. The comparison of the 1D free energy profiles corresponding to the minimum energy paths connecting states A and B in the WT, H163A, and F140A models are shown in (b) . The superimposed structures of the H163A crystal structure against the initial and the final states from the metadynamics simulation of H163A model are described in (d) . In the initial state of the H163A model, the side chain of F140 (green cyan) was present in the ‘in’ conformation whereas, at the end of the simulation, the active site loop was dislocated and the F140 sidechain was exposed to the surface – a conformation similar (light cyan) to that seen in the mutant crystal structure. The sidechain rotation of N28 (purple) is also shown. (f) Free-energy profile for the N28A model shows a “free fall” from state A to state B due to a lack of any significant barrier in its path.

Article Snippet: The H163A mutant was generated in the background of this WT construct (GenScript).

Techniques: Comparison, Mutagenesis